Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

Abstract

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, while less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5.LGR5-positive cells were isolated from 4 adenoma organoid lines and were subjected to RNA-sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA-seq data from adenoma and normal organoids, we found correlations between LGR5 and CRCspecific genes, including DKK4 (dickkopf WNT signaling pathway inhibitor 4) and SMOC2 (SPARC related modular calcium binding 2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Hypothesis: Caco‐2 cell rotational 3D mechanogenomic turing patterns have clinical implications to colon crypts

“…37,38 Readily available biopsy specimens demonstrating distinct epithelial lumen surface Turing patterns can be used to isolate crypts to study stem cells present at the crypt base from healthy normal and abnormal epithelium using in vitro organoid preparations (Figure 2A). 39 We anticipate the programmable "gut-on-a-chip" methodology will demonstrate epithelium morphology similar to the colon lumen surface. This system can be used to study the topology of colon epithelium in response to morphogens including FGF2/glucocorticoids/microbiome, etc.…”

ULCERATIVE COLITIS-DERIVED COLONOID CULTURE: A Multi-Mineral-Approach to Improve Barrier Protein Expression

“…Colonoid cultures were established as described in our recent reports with tissue from healthy subjects (10)(11)(12)(13). Briefly, biopsies were finely minced on ice using a #21 scalpel and seeded into matrigel (Corning), prepared at 8 mg/mL.…”

Calcium-Induced Differentiation in Normal Human Colonoid Cultures

“…Establishment of colonoids from normal human colonic mucosa. Colonoid cultures were established from histologically-normal colon tissue based on our recently described methods (37,39). Briefly, biopsies were finely minced on ice using a #21 scalpel and seeded into matrigel.…”

“…Colonoid culture technology, which is now well-developed (34)(35)(36)(37), provides a way to study human colon polyp responses to potentially useful chemopreventive agents under ex vivo conditions. In a recent study using human colon adenomas in colonoid culture, we found that supplementation of the culture medium with either calcium alone or Aquamin induced differentiation in the adenomas as indicated by a change in morphology and by increased expression of differentiation-related proteins (38).…”